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Membrane Capacitance Recordings Resolve Dynamics and Complexity Of www.nature.com/scientificreports OPEN Membrane capacitance recordings resolve dynamics and complexity of receptor-mediated endocytosis in Received: 22 October 2018 Accepted: 20 August 2019 Wnt signalling Published: xx xx xxxx Vera Bandmann1, Ann Schirin Mirsanaye1, Johanna Schäfer1, Gerhard Thiel1, Thomas Holstein2 & Melanie Mikosch-Wersching1,2 Receptor-mediated endocytosis is an essential process in signalling pathways for activation of intracellular signalling cascades. One example is the Wnt signalling pathway that seems to depend on endocytosis of the ligand-receptor complex for initiation of Wnt signal transduction. To date, the roles of diferent endocytic pathways in Wnt signalling, molecular players and the kinetics of the process remain unclear. Here, we monitored endocytosis in Wnt3a and Wnt5a-mediated signalling with membrane capacitance recordings of HEK293 cells. Our measurements revealed a swift and substantial increase in the number of endocytic vesicles. Extracellular Wnt ligands specifcally triggered endocytotic activity, which started immediately upon ligand binding and ceased within a period of ten minutes. By using specifc inhibitors, we were able to separate Wnt-induced endocytosis into two independent pathways. We demonstrate that canonical Wnt3a is taken up mainly by clathrin-independent endocytosis whereas noncanonical Wnt5a is exclusively regulated via clathrin-mediated endocytosis. Our fndings show that membrane capacitance recordings allow the resolution of complex cellular processes in plasma membrane signalling pathways in great detail. Wnt signalling is a highly-conserved signalling pathway with important functions in development, tissue-homeostasis, stem cell biology and many diseases, including cancer. Afer three decades of dedicated research we have come to understand many of the fundamental components of Wnt signalling pathways. However, it remains puzzling how so many diferent processes may be regulated by only one system. Wnt signalling in principle is highly complex because it is mediated by the interplay of a wide variety of ligands, receptors, co-receptors, antagonists, agonists and intracellular factors, which are deeply embedded in metazoan genomes1–4 and interact in a distinct manner. Te secreted Wnt proteins activate diferent downstream pathways, which are traditionally classifed as canonical (ß-catenin dependent) and noncanonical (ß-catenin independ- ent). In the canonical Wnt pathway, binding of the extracellular Wnt ligand to the Frizzled receptor leads to the formation of a complex with the co-receptor Lrp5/6. Tis complex recruits the scafolding proteins Dishevelled and Axin, as well as GSK3 and several other intracellular components, to build up the Lrp6-signalosome. Upon formation, the Lrp6-signalosome inhibits the phosphorylation of ß-catenin, thus marking it for proteasomal degradation. As a result, there is stabilization of ß-catenin in the cytosol, which regulates various Wnt target genes upon translocation into the nucleus5. In contrast, the noncanonical Wnt/PCP pathway utilises Frizzled receptors to activate Dishevelled and regulates various downstream efectors such as small GTPase’s of the Rho and Rac subfamilies, the CaMKII and the PKC pathway6. Cellular mechanisms add another layer of complexity to Wnt signalling, for instance endocytosis of the activated receptors7,8. Recent reports show that Wnt signalling can be inhibited when endocytosis of the ligand-receptor complex is blocked9–16. Thus, endocytosis is not only necessary for the degradation of ligand-receptor complexes but also crucial for signal activation. Te underlying mechanism for this is, however, unclear. Tere may be several explanations, e.g. the ligand-receptor complex might require acidifcation in the 1Department of Biology, Technische Universität Darmstadt, Schnittspahnstrasse 3, 64287, Darmstadt, Germany. 2Centre for Organismal Studies, University of Heidelberg, Im Neuenheimer Feld 230, Heidelberg, 69120, Germany. Correspondence and requests for materials should be addressed to M.M.-W. (email: [email protected]) SCIENTIFIC REPORTS | (2019) 9:12999 | https://doi.org/10.1038/s41598-019-49082-4 1 www.nature.com/scientificreports/ www.nature.com/scientificreports endocytic vesicles for activation or internalisation is required for the interaction with cofactors. Nevertheless, the mode of uptake of Wnt ligands bound to their receptor is still unknown. Despite this gap in basic knowledge, a wealth of data has demonstrated distinct diferences in the endocyto- sis of canonical and noncanonical Wnt signalling. Both clathrin-mediated endocytosis and caveolin-dependent endocytosis are involved in canonical Wnt signalling17. After formation of Lrp6-signalosomes in response to Wnt3a, these are internalised through a caveolin-mediated route12,18–20 but Wnt3a has also been shown to trigger clathrin-mediated endocytosis9,11,21,22. Endocytosis of the canonical Wnt8 induced Lrp6-signalosome via clathrin-mediated endocytosis has been discovered in zebra fish15. For the noncanonical Wnt5a, only clathrin-mediated endocytosis has been reported23–27. Ohkawara et al.28 demonstrated that clathrin-dependent endocytosis of Wnt5a is essential for Wnt/PCP-signalling in Xenopus. Knowledge of receptor-mediated endocytosis following Wnt stimulation is so far restricted to insights from microscopic and/or biochemical analyses. However, these approaches are indirect and limited to the investigation of the endpoint of endocytosis. Tey lack information on the primary process of endocytosis and fail to provide insights into the dynamics and the temporal resolution of the endocytic process. However, endocytosis may be monitored with high spatial and temporal resolution with recordings of the membrane capacitance29,30. In fact, the level of detail provided by membrane capacitance measurements is sufcient to analyse individual vesicle fssion events on the plasma membrane in living cells. Te method takes advantage of the fact that exo- and endo- cytosis are associated with changes in plasma membrane area, which, in turn, generate proportional changes in the electrical membrane capacitance (Cm). Tus, even the fssion and fusion of single endo- and exocytic vesicles can be resolved in real time in single living cells31. Here, we employed the membrane capacitance technique to resolve Wnt receptor-mediated endocytosis with high temporal and spatial resolution in real time. We were able to demonstrate that challenging of HEK293 cells with a canonical (Wnt3a) and a noncanonical ligand (Wnt5a) triggers an immediate increase in endocytosis of small vesicles and that both ligands use separate endocytic pathways; while Wnt5a is endocytosed by clathrin-coated vesicles, Wnt3a takes a clathrin-independent endocytic pathway. Tus, our data provide the frst direct measurements of endocytotic processes in Wnt signalling on the cellular level. Results Characterisation of receptor-mediated endocytosis of Wnt3a and Wnt5a in whole cell mode in HEK293 cells. We used recombinant human canonical Wnt3a and noncanonical Wnt5a protein to analyse their endocytic uptake into HEK293 cells as a model system. Tese cells contain the Wnt signalling machinery endogenously and exhibit a robust Wnt response18,32,33. In a frst assay, HEK293 cells were incubated with an endocytosis marker, the styryl dye FM 4-64 (10 µM) and subsequently challenged with either recombinant Wnt3a or Wnt5a (5 ng/ml). Te confocal data in Fig. 1A show that unstimulated HEK293 cells exhibited a constant rate of endocytosis. Addition of Wnt3a or Wnt5a to the bath strongly increased endocytosis. While the response to Wnt3a was immediate, Wnt5a-stimulated endocytosis seemed to occur only afer a lag of fve minutes. Afer 30 minutes, it even exceeded the amount of endocytosis in Wnt3a-treated cells (Fig. 1B). Te stimulated uptake of FM 4-64 dye suggests that Wnt ligands induced endocytosis as an early step in the signal transduction cascade. Motivated by these fndings, we next analysed the rapid efect of Wnt ligands on endocytosis by whole-cell patch-clamp capacitance measurements. Figure 1C shows a representative recording of an unstimulated HEK293 cell. Tese cells typically exhibited a stable capacitance of 4–15 pF. Tis corresponds to a membrane area of 500–1875 µm2 (diameter of 12–24 µm), an area typical for HEK293 cells. Te record- ings show that the Cm value remains mostly constant over the time of recording under control conditions. Tis implies that endocytosis (as visualised by the uptake of FM 4-64 dye in Fig. 1A) was balanced by exocytosis and thus a constant cell surface was maintained. Te representative recordings in Fig. 1C show that exposure of the cells to Wnt3a and Wnt5a (5 ng/ml) in the bath solution caused an immediate and continuous decrease in the Cm value, indicating that both Wnt ligands stimulated endocytosis. In all tested cells, the Cm value dropped within one minute of stimulation by about 0.5 pF (±0.29 pF). Considering a specifc membrane capacitance of 0.8 µF/ cm2, this translates into a decrease in membrane area by 62 µm2 (±36 µm2) (diameter 4.5 µm ±3.4 µm). Figure 1D summarises the mean time course of ligand-stimulated endocytosis over ten minutes. Both ligands triggered a decrease in capacitance signifcantly below that of the background signal in control cells. Te data further show that the ligand-stimulated increase in endocytosis approached saturation already about three minutes afer stim- ulation. Furthermore, Wnt3a induced a maximal drop in the Cm values (2.2
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